Cytomegalovirus
Cytomegalovirus (CMV) is an important human pathogen and a major opportunist, which emerges to cause disease in the immuno-compromised such as AIDS patients, neonates, and individuals who have been given immunosuppressive drugs as part of a transplantation regimen. In these individuals, the consequences of CMV in acute or re-emerging infections can be dire, including retinitis, encephalitis, and pneumocystis, among other pathologies. Furthermore, in immuno-competent hosts, CMV establishes a persistent lifelong infection through which it has been linked to a variety of inflammatory conditions including coronary artery occlusion following heart transplant and atherectomy and restenosis following angioplasty. CMV interacts with leukocytes during acute infection of the host as well as during lifelong latency. As such, leukocytes are important players in CMV-induced diseases and have been implicated in the acute phase of infection as vehicles for dissemination of virus and as sites of residence during lifelong latency.
CMV infection affects approximately 30 to 60 percent of the estimated 29,000 patients receiving bone marrow or solid organ transplantations in the US annually, causing transplant rejection, serious illness and even death if untreated. Expensive antiviral drug therapy is used to control the disease, but does not eliminate the infection. These treatments cost per patient between $30.000 to $50.000 USD a year. CMV infection causes severe consequences in about 3,600 infants and death in about 400 each year in the U.S. CMV infection also affects HIV/AIDS patients, with an estimated 40% of all AIDS patients requiring treatment against CMV infection. There currently remains no effective cure for CMV infection. Viral suppressants do exist, however, carry strong side effects and serve only to control infection.
The most common drugs for the treatment of CMV infection in transplantation patients and HIV/AIDS patients are the generic drugs Ganciclovir and Acyclovir, originally developed for herpes simplex virus (HSV). Ganciclovir and Acyclovir have a suppressing effect on CMV as well as on HSV. Vistide from Gilead is a newer compound that is expected to grow and take over the market with Roche's Valcyte at the expense of the older generic drugs.
None of the existing drugs, patented or generic, can eradicate the infection, merely halting the CMV disease progression in immuno-compromised or immuno-suppressed patients. In recent clinical studies, Foscavir and Ganciclovir were compared on their ability to treat immuno-compromised patients. The results showed a 30% better suppression of the infection using Foscavir. However 40% of the patients on Foscavir later switched to Ganciclovir because of intolerable nausea. These results show that there is room for improvement both in efficacy and in toxicity levels.
Immunotoxins
An immunotoxin is a ligand combined with a toxin, which can be used to kill cells expressing receptors for the ligand. Immunotoxin treatment is also known as ligand-targeted therapeutics. Thus, the immunotoxins contain a targeting moiety (a ligand) for delivery and a toxic moiety for cytotoxicity. The ligands currently used are monoclonal antibodies, cytokines/growth factors and soluble receptors. An advantage with immunotoxins over e.g. traditional chemotherapy drugs is, that the cells need not be dividing to be killed. Furthermore, if the immunotoxin is efficiently internalized, side effects will not occur in antigen negative cells.
In general, however, immunotoxins have not shown impressive levels of efficacy. A common problem is that they are not sufficiently specific for the diseased cells, and furthermore, often are incapable of efficiently entering the diseased cells to exert its cytotoxic effects. Immunotoxins also result in higher levels of systemic toxicity than other therapies, presumably because of non-specific uptake of the immunotoxin.
Currently, new approaches to immunotoxins are being explored to overcome problems of toxicity, immunogenicity, and heterogeneity of antigen expression. These approaches include the use of genetic engineering to fuse the translocation and catalytic domains of toxins to human single chain antibodies and to use phage display to select high affinity, tumour-selective ligands. Use of bivalent constructs can also increase the affinity and potency. Other approaches, centres around the selection of ligands that target tumour vascular endothelium and the targeting of oncogene products or differentiation antigens. In spite of that research on immunotoxins has been ongoing in the last two decades, no immunotoxin against virus related diseases is available on the market.
Immunotoxins tend to be more useful in haematological malignancies, which are characterized by a high percentage of malignant cells that express the target antigen in contrast to solid tumours, which are characterized by a mixed cell population, and cells that are often not easily accessible for the immunotoxin. In the case of targeting tumour cells, monoclonal antibodies that also target normal cells are typically used because unique tumour associated antigens have not been identified on most tumour cells. Even though the tumour cells express higher levels of the selected antigen, and tumour cells are preferentially killed, the treatment is still often associated with significant side effects. Another drawback has been the mouse origin of the monoclonal antibodies, which are immunogenic in humans. This problem has been largely solved by the use of human antibodies and also by using recombinant human growth factors, which are not immunogenic in humans.
Immunotoxin Internalization
Most immunotoxins are developed against cells that have undergone malignant transformation and as a part of this transformation therefore overexpress a certain antigen or a group of certain antigens. Even though these antigens are overexpressed on the transformed cells, they are rarely specific for the transformed cells, but are often also expressed on normal cells. Thus, only a few cellular antigens are over expressed on transformed cells. Therefore, to avoid undesired toxicity by killing normal cells expressing the target antigen, drug developers are restricted to target very few candidate disease antigens, and drug developers have therefore traditionally been restricted to select a target antigen solely based on the cell type distribution. Consequently, many immunotoxins have not been able to efficiently enter the target cells, even though they bind the target antigen with high affinity, resulting in inadequate potency.
One attempt to solve the problem of getting the immunotoxin efficiently into the target cell has been made by He D. et al, 2005. They used arginine-containing membrane translocation signals (MTS) (e.g. Tat and VP22) as carriers for transporting an arginine (Arg9-peptide) containing immunotoxin, PE35/CEA(Fv)/KDEL into carciniembryonic antigen (CEA) expressing target cells. The authors suggests, that incorporation of the Arg9 peptide (a 9-mer arginine peptide) into the immunotoxin facilitates the receptor-mediated endocytosis of the PE35/CEA(Fv)/KDEL immunotoxin. Unfortunately, the introduction of a MTS signal in the immunotoxin result in loss of specificity towards cells expressing the target antigen. Therefore this approach seems not to be the solution to the problem of how to get the immunotoxin into the target cells.
Immunotoxins consisting of a cytokine/growth factor and a toxin (a cytokine-based immunotoxins) has the advantage that it can be effectively internalized after cytokine mediated receptor activation followed by receptor internalization. However, cytokine based immunotoxins suffers from two major problems. 1) Cytokine-induced receptor internalization requires that the ligand retain agonistic properties, which may have unwanted stimulatory effects on the target cells and other cells bearing the receptor. 2) Cytokine-based immunotoxins interacts with cytokine receptors on normal cells and thereby kill normal cells in addition to the diseased target cells. Thus a successful cytokine based immunotoxin need 1) to be able to stimulate internalization without retaining agonistic properties that may induce unwanted stimulatory effects, and 2) to interact and be internalized in diseased cells only and not by healthy cells bearing the same cytokine receptor.
Immunotoxins for Anti-Viral Therapy
HIV
Immunotoxins have been evaluated for therapy of HIV infection. For example, immunotoxins have targeted to CD4, with some success. Also soluble CD4 has been conjugated to toxins, and the resulting immunotoxin inhibits synthesis of viral proteins in infected cells and spread of virus in vitro. Immunotoxins have also been targeted to the HIV envelope protein gp160 and it's components gp120 and gp41. However these attempts have had little effect, partly because of HIV antigenic variation and partly because of anti-epitope antibodies present in the serum of infected individuals.
CMV
Prior attempts to treat CMV using immunotoxin strategies have not been successful.
An academic research group has prepared and tested immunotoxins targeting CMV-infected cells (Barnett et al. 1995, Smee at al. 1995, Barnet et al, 1996). Barnett et al. (1995) described the generation of an immunotoxin specific for cells infected with human CMV, and an immunotoxin specific for cells infected with mouse CMV. Both immunotoxins were polyclonal, i.e. the antibodies were not directed against a specific well defined target antigen. Furthermore, the anti serum (anti MCMV and HCMV) were only purified by protein A affinity, thus the purified polyclonal antisera contains a non-defined pool of antibodies, against CMV antigens (MCMV or HCMV) and against other non-defined antigens. The authors state that “The virus specific antibody in these preparations accounted for less than a few percent of the total IgG.” The polyclonal antibody pool (antisera) were coupled to the toxin gelonin. The effect of the anti human CMV immunotoxin were measured by the ability of the immunotoxin to inhibit s35-metheonine incorporation into proteins as a measure of the immunotoxin ability to inhibit ribosome activity. The authors show no data on the ability of the anti human CMV immunotoxin to inhibit the growth or replication of human CMV in infected cells. More over the authors show, that the anti human CMV immunotoxin only inhibits 35S-methionine incorporation by approximately 15%. In contrast, the authors show that the anti mouse CMV immunotoxin can inhibit the incorporation of S35-metheonine >90%. Additionally, the authors state that addition of the anti mouse CMV immunotoxin to mouse cells (mouse mammary tumor cell line, C127I) infected with mouse CMV at an MOI of 0.001 inhibited the virus yield at 7 days post infection with approximately 2 log (100 fold) at 20 μg immunotoxin/ml.
Three papers describe the use of monoclonal antibodies against mouse cytomegalovirus (Smee et al. 1995a, Smee et al. 1995b, Barnett et al. 1996). The antibodies were generated from mouse CMV infected BALB/c mice. Treatment of MCMV infected cells with the monoclonal antibody D5.F10.B8 (not coupled to any toxin) caused a 3-3.5 log 10 decrease in virus titer, but there was no dose response effect among the various concentrations tested (1.25, 2.5, 5, 10 and 20 μg/ml) (Smee et. al. 1995a). However the authors show a synergistic inhibition of virus yield using a combination of high concentrations of monoclonal antibody in combination with either high concentrations of ganciclovir or high concentrations of (S)-1-[3-hydroxy-(2-phosphonylmethoxy)-propyl]cytosine (HPMPC). In vivo, treatment with the monoclonal antibody alone or in combination with either 25 or 50 mg/kg/day ganciclovir has no or only minor effect in MCMV induced mortality in SCID mice.
Coupling of the neutralizing monoclonal antibody D5.F10.B8 or of the non-neutralizing monoclonal antibody C34.18.F6 to recin A chain generated two antibody based immunotoxins against mouse CMV. It should be noted that even thought the antibodies are monoclonal, the antigens are not defined. Treatment of MCMV infected cells with the monoclonal immunotoxins caused a 2-3 log 10 decrease in virus titers. Additionally, the authors show a synergistic inhibition of virus yield using a combination of high concentrations of monoclonal antibody in combination with either high concentrations of ganciclovir or high concentrations of (S)-1-[3-hydroxy-(2-phosphonylmethoxy)-propyl]cytosine (HPMPC). In vivo, treatment with the immunotoxins alone had no effect on MCMV induced mortality in SCID mice. Combination therapy with either the D5.F10.B8 or the C34.18.F6 based immunotoxins with 50 mg/kg/day ganciclovir appeared to suggest a mild synergy in delaying MCMV induced mortality in SCID mice from 27.3±2.5 (ganciclovir alone) to 29.3±1.1 days (ganciclovir+C34 immunotoxin) and 30.4±3.1 days (ganciclovir+D5 immunotoxin) (Smee et al. 1995b). The authors explain the pour effect of the immunotoxins with lack of knowledge on the actual concentrations of immunotoxin in the animals treated, and lack of knowledge on whether the immunotoxins reached the site of viral replication within the animal. Furthermore the authors do not show any data on the actual target antigen, whether the antigen is expressed in the infected animals, the kinetics of the immunotoxin in the animal, the affinity of the immunotoxins to the infected cells or whether the antibodies are internalized into the infected cells. Also, the authors show no difference in the effect on viral infection between the unconjugated monoclonal antibodies or the toxin conjugated antibodies.